Alkylidine and arylidine amino acid esters, and alkyl and aryl amino acid esters

ABSTRACT

Several series of new organic compounds and methods of preparing same are disclosed. These include compounds of the general formula for alkylidine and arylidine amino acid esters: AND THE GENERAL FORMULA FOR ALKYL AND ARYL AMINO ACID ESTERS: For both these formulas, R1 and R3 are each of the group consisting of H, an aryl or alkyl radical, with or without other functional groups and R2 is an alkyl radical or aryl radical with or without functional groups. The compounds are useful for identification of amino acids by gas liquid chromatography and as pharmaceuticals, and for the separation of L-amino acids from natural sources, such as protein and peptide hydrolysates.

United States Patent 91 Davis [451 Feb. 27, 1973 ALKYLIDINE ANDARYLIDINE AMINO ACID ESTERS, AND ALKYL AND ARYL AMINO ACID ESTERS [76]Inventor: Jefferson W. Davis, c/o Radio-Carbon Lab., 1012 WashingtonStreet, San Carlos, Calif. 94070 [22] Filed: Oct. 18, 1967 211 App].No.: 676,057

[52] US. Cl. ...260/482 R, 260/326.l4 T, 260/326.3, 260/471 R, 260/481R, 260/482 P [51] Int. Cl. ..C07c 101/14 [58] Field of Search ..260/47lA, 482 R, 326.14 T, 260/3263, 471 R, 481 R, 482 P [56] 1 ReferencesCited UNITED STATES PATENTS 3,361,626 H1968 Harris et a]. ..260/482 R3,419,525 12/1968 Aelony ..260/482 R OTHER PUBLICATIONS Fieser, L. F. etal., Organic Chemistry, 1956, pub. by Reinhold Pub. Corp. of New York,pages 211 to 214.

Primary Examiner-Henry R. Jiles Assistant Examiner-L. Arnold ThaxtonAttorney-Julian Caplan [57] ABSTRACT Several series of new organiccompounds andmethods of preparing same are disclosed. These includecompounds of the general formula for alkylidine and arylidine amino acidesters:

and the general formula for alkyl and aryl amino acid esters:

3 Claims, No Drawings ALKYLIDINE AND ARYLIDINE AMINO ACID ESTERS, ANDALKYL AND ARYL AMINO ACID ESTERS The present invention relates tocompounds of the general formulas set forth in the abstract ofdisclosure. The invention further relates to methods of preparing suchcompounds and also for separating mixtures of amino acids by firstpreparing such compounds, fractionally distilling same and thenobtaining free pure amino acids from the distillates.

Gas liquid chromatography identification of amino acids has heretoforebeen a complex and time-consuming operation because of difficulties withvolatility and separability of derivatives when such acids have reactedwith other reagents. The esters of the present invention, however, haveunusually sharp boiling ranges and show no sign of decomposition duringchromatographic analysis. Accordingly, a principal function of thepresent invention is in the identification of amino acids for chemicalresearch, pharmacology, interstellar investigation, and many otherpurposes.

Still another feature of the invention is in the manufacture of pureamino acids. Protein hydrolysates containing pluralities of amino acidsand derivatives are converted to mixtures of the compounds of thepresent invention. Because of the sharp boiling point ranges of thesecompounds, fractional distillation of the mixtures yields quite pureester compounds. The latter are then treated with acids or bases toyield amino acids in relatively free form. Such separation has notheretofore been accomplished in such rapid and economical manner.

The foregoing and other objects-and advantages of the invention will beapparent to those skilled in this art by consideration of the followingexamples:

EXAMPLE 1 To an amino acid methyl ester was added an aldehyde. Themixture was dissolved in a non-polar solvent containing a drying agentand the drying agent separated by filtration. The product was distilledto yield an alkylidine amino -=acidester, a Schiff base. Morespecifically, at room temperature and atmospheric pressure, to 17 parts(by weight, throughout this specification) of leucine methyl ester wasadded ten grams isobuteraldehyde, with stirring. The mixture became warmand small droplets of water formed. A non-polar solvent, such asmethylene chloride, was added together with a drying agent, such as Na,Mg or Ca sulphate in sufficient quantity to remove the water formed.After standing a few minutes, the solution was separated by filtration,concentrated at a reduced pressure, such as mm., and distilled to give19.1 grams of isobutylidine leucine methyl ester, a colorless oil boil-.

ing at l03lO4C. at 15 mm. pressure.

By using a corresponding quantity of other appropriately esterifiedamino acid in place of leucine methyl ester, the same method has beenused to produce the designated products in Table I. Additional arylidineproducts are shown in Table II. It will be understood, of course, thatthe aldehyde was appropriately varied in instances where other than theisobutylidine product is shown.

The amino acid ethyl esters when reacted with an aldehyde yield morestable compounds than the corresponding methyl estersfThe amino acidethyl esters are thus useful in gas liquid chromatography. Morespecifically, alanine ethyl ester was treated in the same manner as theleucine methyl ester of the preceding example to produce isobutylidinealanine ethyl ester. Gas liquid chromatography of the product yielded asharp peak and thus the product is useful as a standard in gas liquidchromatography of amino acids.

The ilidine amino acid methyl esters tend to decompose in some of thecommercially available gas liquid chromatogrophy equipment and hence itmay be desirable to reduce the ilidine compounds to the alkyl and arylamino acid esters, as in Example 2, prior to chromatography. However, inchromatography equipment where metal is not used in construction orwhere the metal does not tend to catalyze the decomposition of themethyl esters of the amino acids, then ilidine compounds of amino acidmethyl esters may be used directly in gas liquid chromatography.

The same method was used in the other esters of amino acids.

EXAMPLE 2 The compounds of Example 1 were reduced. As a first step theywere dissolved in an alcohol corresponding to the ester function inwhich was suspended zinc dust or tin metal, stirred and held at reducedtemperature and HCl added until the zinc (or tin) was dissolved. Thesolution was concentrated to a syrup, cooled, and concentrated ammoniaadded. The liberated oil was extracted into a non-polar solvent. Theproduct was fractionally distilled to yield an alkyl or aryl amino acidester.

More specifically, at room temperature and atmospheric pressure, tenparts of isobutylidine leucine methyl ester was dissolved in 200 partsof absolute methanol in which was suspended 20 parts of zinc dust or tinmetal. (Where an ethyl ester was used, the alcohol was ethanol, andsimilar corresponding alcohols used for the various esters). Whilestirring vigorously, the temperature was held below 10 C. by use of iceor other cooling means, and dry HCl or l-lBr was passed into thesolution until all zinc or tin had dissolved. The solution wasconcentrated to a syrup at reduced pressure, such as 15 mm., and about35 C. The syrup was treated with ice and concentrated ammonia, and theliberated oil extracted into a solvent such as methylene chloride, ethylether or other non-polar solvent. After removal of the solvent andfractionation of the residue, there was obtained 8.2g. of N-isobutylleucine methyl ester which boiled at lOl-l02 C. at 15 mm. pressure.

Gas liquid chromatography of the foregoing gave very sharp peaks. Thecompounds are therefore useful as standards in the gas liquidchromatography of amino acids.

By methods similar to the above and by starting with a correspondingquantity of other appropriately esterified amino acid and the alcoholcorresponding to the ester function, there have been produced thedesignated alkyl and aryl products listed in Table I.

These compounds likewise produced excellent peaks in gas liquidchromatography.

The ilidine compounds when reduced in the presence of an excess ofaldehyde gave dialkyl or diary] or arylalkyl amino acid esters. Thelatter are also useful in gas liquid chromatography.

EXAMPLE 3 The compounds of Example 2 were dissolved in alcoholcontaining base or acid, then neutralized to give the free amino acidderivatives. More specifically, 5 parts N-isobutyl leucine methyl esterwas dissolved in 25 parts of ethanol or other alcohol containing aslight excess (e.g. 26 parts) of either a base, such as NaOH, or an acidsuch as HCl, HBr or H 50 and the mixture warmed on a water bath to atemperature of about 80 C. for about 15 minutes. The solution was thenmade neutral and the white solid, N-isobutyl leucine, having a MP. of260-262 C, was obtained.

The method is also useful for production of other N- alkyl or N-arylamino acids.

EXAMPLE 4 A protein hydrolysate containing a mixture of amino acidderivatives was esterified and then processed substantially as inExample 1. The mixture of esters was fractionally distilled to separateout the various amino acid derivatives present.

More specifically, a protein such as 100 parts gelatin was converted tothe free amino acid hydrochlorides by boiling for about 12 hours with375 parts of approximately eight normal aqueous hydrochloric acid. Thehydrolysate was treated with charcoal and filtered and the filtrateevaporated to dryness under a reduced pressure, such as 15 mm. Theresidue was suspended in 500 parts of methanol or other suitable alcoholand saturated with dry hydrogen chloride or hydrogen bromide. Thesolution was heated on a water bath at about 60 C. for about one hourand the alcohol removed by evaporation at about 15 mm. The residue wasthen suspended in 1000 parts methylene chloride or other chlorinatedsolvent and with vigorous stirring and cooling to about C. saturatedwith anhydrous ammonia. The ammonium chloride was removed by filtrationand the filtrate concentrated at mm. The remaining oil was treated with125 parts of isobutyraldehyde. After a few minutes 500 parts ofmethylene chloride or ether or other non-polar solvent was added, alongwith approximately 60 parts of sodium sulphate. After filtration thesolution was concentrated and fractionally distilled at about 0.1 mm.pressure.

Accordingly, the derivatives of the various amino acid derivatives ofproteins may conveniently be separated. Each such derivative may then beconverted to other compounds of the original acid, as by Example 6.

EXAMPLE 5 A mixture containing ten grams each of the methyl esterhydrochlorides of alanine, glycine, leucine, isoleucine, valine,threonine, serine, glutamic acid, aspartic acid, tyrosine, tryptophan,arginine, lysine, ornithine, proline, hydroxyproline, cysteine, cystine,methionine and phenylalanine was treated with sodium hydroxide until thesolution was basic, while keeping the solution between 0 and l0 C. Otherorganic or inorganic bases such as potassium hydroxide, ammonia,pyridine quinoline or triethyl or trimethyl amine may be used. The coldsolution (15 to C.) was extracted thoroughly with isobutyraldehyde, 3 to4 times with 300 C. each time. The isobutyraldehyde extract was driedover sodium sulphate, and after filtration the excess aldehyde removedunder reduced pressure, 15 mm., (water pump). The residue wasfractionated to give the isobutylidine methyl esters of the followingamino acids in 60-80 percent yields:

Alanine, B.P. 7475 C. 15 mm;

Glycine, B.P. 7778 C. 15 mm;

Valine, B.P. 9293 C. 15 mm;

Leucine and lsoleucine, B.P. l03-l05 C. 15 mm;

Serine and Threonine, 64-65 C. 0.1 mm;

Cysteine, B.P. 73-74 C. 0.1 mm;

Methionine, B.P. 80-8 1 0.1 mm;

Aspartic acid, B.P. -86 C. 0.1 mm;

Glutamic acid, B.P. l01 C. 0.1 mm;

Phenylalanine, B.P. Illl 12 C. at 0.1 mm;

Lysine (mono) and Ornithine (mono), l24-l27 C.

at 0.1 mm.

Proline, hydroxyproline, cystine and arginine react only on long contactwith the aldehyde and were recovered from the extracted water solutionas the free acids by conventional methods. Tyrosine, B.P. l76l7 7 C. 0.1mm. and Tryptophan, B.P. 189l90 C. 0.1 mm. were recovered from the potfrom the distillation by short path distillation in yields of about 50percent. No histidine was recovered.

The operating procedures were as follows:

Procedure A. In water solution: The ester salts were poured over crushedice and the base added until pH8 was reached. The extraction procedurewas carried out at once.

Procedure B. In organic solution: The ester salt was poured into asuitable solvent and a mixture of the amine and aldehyde added all atonce. After standing for a few minutes the solution was washed withwater and the organic layer dried over sodium sulphate or other dryingagent. After separation from the drying agent, excess aldehyde wasremoved under reduced pressure and the residual ilidine estersdistilled. The results were essentially the same in both cases.

EXAMPLE 6 The arylidine or alkylidine amino acid esters were convertedto the free amino acids and the aldehyde as follows by using base oracid:

The alkylidine or arylidine amino acid ester was treated with acid andthe mixture steam distilled until no more aldehyde passed over. Uponevaporation of the residue the pure amino acid salt was obtained. Thefree amino acid was obtained from these salts by conventional means.More specifically:

a 10 parts of isobutylidine glycine methyl ester was dissolved in watercontaining 10 parts of hydrochloric acid and steam passed through thesolution until isobutyraldehyde no longer passed over. Upon evaporationof the residue, glycine hydrochloride was obtained in quantitativeyield. The same was accomplished by boiling the mixture for half anhour, removing the aldehyde by extraction or distillation andevaporation of the residue to dryness. The free amino acid salts of allthe other derivatives were obtained in a similar manner.

b. 10 parts of isobutylidine glycine methyl ester is dissolved in watercontaining 10 parts of NaOH (or KOH etc.) and the solution boiled forhalf an hour. The aldehyde was removed and the water solution containingthe salt of the amino acid was made neutral and the amino acid isolatedby conventional means.

EXAMPLE 7 The esters of Example I may be converted to the N- isobutylcompounds of amino acids. More specifically, 5 parts of isobutylidinevaline methyl ester was dissolved in 100 parts of absolute methanol, orother alcohol, in which is suspended a catalyst, such as 0.2 parts ofplatinum (or other noble metal) oxide. The mixture was hydrogenated at45 psi until no more hydrogen was absorbed. The catalyst was removed byfiltration, the solution concentrated under a reduced pressure, such asmm., and the oil fractionated to give 4. lg of N-isobutyl valine withB.P. 77-78 C at 15 mm. pressure. The same method was used to producecorresponding compounds of the other amino acids, with the exception ofcysteine and methionine (i.e. amino acids which contain sulphur andaffect the catalyst).

EXAMPLE 8 This example used a'catalyst different from that of Example 7,but is in other respects similar. More specifically, 5 parts ofisobutylidine valine methyl ester was dissolved in 100 parts of absolutemethanol or other alcohol, in which was suspended 7 parts of freshlyprepared Raney nickel. The mixture was hydrogenated at 45 psi until nomore hydrogen was absorbed. The catalyst was removed by filtration, thesolution concentrated under reduced pressure and the oil fractionated togive 3.8g of N-isobutyl valine with B.P. of 77-78 C. at 15 mm. pressure.The same method was used to produce corresponding compounds of the otheramino acids, including cysteine and methionine.

EXAMPLE 9 Reduction of the ilidine compounds without use of a catalystmay be accomplished with use of formic acid. For example, 10g ofisobutylidine valine methyl ester was dissolved in g. of 99 percentformic acid and the mixture was heated to 7585 C. until evolution of COceased. Upon fractionation of the mixture, isobutyl valine methyl esterwas obtained in a yield of over 90 percent. Other ilidine esters gavesimilar results.

' EXAMPLE 10 EXAMPLE 1 l A convenient, more direct preparation of alkylor aryl amino acid esters employed the acid salts of the amino acidesters, to which were added crushed ice, the aldehyde selected and anon-polar solvent, such as ether, followed by rapid addition of a strongbase until the solution was slightly basic. The organic layer wasremoved and dried. The solvent and excess aldehyde was removed bydistillation to give the desired product.

More specifically, to 10g. glycine methyl ester hydrochloride was added50g. crushed ice, 15 ml. isobutyraldehyde and 100 ml. ether followed byrapid addition of 10 percent NaOH until the solution was slightly basic,pI-l8 indicator being added to the acid solution. After shakingthoroughly the organic layer was removed and dried over sodium sulphate.Upon removal of the ether and excess aldehyde under reduced pressure,the product distilled to yield 10g isobutylidine glycine methyl esterboiling at 77-78 C. at 15 mm.

Alternatively, 10g glycine methyl ester hydrochloride was dissolved in50 ml methanol at l0 C. containing an equivalent amount of sodiummethoxide, and the 15 ml isobutyraldehyde, ether and sodium sulphateadded. The mixture was then filtered, concentrated and distilled. Yieldsof above 90 percent were obtained.

It will be understood that other amino acid esters and other aldehydesmay be used in this example.

EXAMPLE 12 This example illustrates manual or automatic methods of gasliquid chromatography using the alkylidine or arylidine derivativeswhich have been described in certain of the preceding examples. It willbe understood that the following example is subject to considerablemodification, as will occur to one skilled in the art.

A water solution containing a mixture of amino acid and/or amino acidderivatives was treated with an ion exchange resin in the acid form. Thewater was replaced by an alcohol, such as methanol, producingcorresponding amino acid esters. The excess alcohol -was removed and themixture treated with an amine and an aldehyde to yield an ilidinecompound, which was dried, freed from the resin and excess reagents andthen reduced to the corresponding alkyl or aryl amino acid esters.

More specifically, lg. of glycine in water solution was placed on acolumn containing a sulphonic acid resin in the acid form. The aminoacid remained on the column, all acidic impurities passing through. Thecolumn was then treated with methanol and heated, forming the amino acidester of glycine. A mixture of isobutyraldehyde and triethyl amine waspassed through the column, removing the amino acid ester as theisobutylidine ester, identical to those described in the other examples.

TABLE I PHYSICAL PROPERTIES OF ISOBUTYLIDINE AMINO ACID METHYL ESTERSAND THEIR lsoleucinc I) 104-105 89 2) 96-97 a-Amino- 1 90-91 96 butyricAcid 2) 85-86 Methionine 1) 144-145 93 2) 78-79(01) Valine 1) 92-93 752) 77-78 Serine l) 65-66(01) 84 2) 62-63(01) Threonine 1) 64-65(01) 962) 66-67(00) Phenylalanine 1) 111-112(0.l) 97 2) 113-114(01)Lysine(mono) 1) 124-125(0.l) 50 2) 119-120(O.1) Ornithine l) 126-127(01)60 (mono) 2) 118-120(0.1) hydroxyproline 1) 112-114(0.1) 2) 110-111(0.1)Tryptophan 1) 192-l93(0.1 88

2) 189-190(01) Cysteine 1) 73-74(0. 1) 76 2) 76-77(0.1) Aspartic acid 185-86(0. 1) 88 2) 75-76(0.1) Glutamic acid I) l-10l(0.1) 96 2) 91-92(01)Proline 1) 88-89(01) 50 Benzylidine valine methyl ester 119-120(01) 95N-Benzyl valine methyl ester 96-97(0. 1 85 lsobutylidine leucinesec-butyl ester Tyrosine 13.75( 103 C..H1:, 1 IO ZI Z u m z zi CHHIUOHN(:1) Boiling points are uncorrected and determined at 15 mm pressure(water pump) unless otherwise noted. First b.p. given is for ilidinecompound, second is for N-alkyl or N-aryl compound.

(b) Yields given for ilidine compound only.

(c) Column used was Carbowax 0.02 in. X ft. capillary. Carrier gas, N:at 6 ml/mi. Program: isothennal at temperatures shown. Instrument:Perkin-Elmer 800 Series Gas Chromatograph. Retention time in minutes.

TABLE [1 Boiling Points of Benzylidine Amino Acid Esters Amino AcidB.P.C at 0.1 mm pressure Norleucine 123-124 Alanine 114-115 Norvaline123-124 lsoleucine 1 19-120 Methionine 164-165 Serine 138-139 Proline133-134 Aspartic acid 149-150 Valine 119-120 Phenylalanine -176Thrconine 135-136 What is claimed is: l. A compound of the formula,

2. Isobutylidine alpha amino acid esters wherein said ester function isof the group consisting of methyl, ethyl and butyl. 3.Trichloroethylidene valine methyl ester.